Recently, the nanotechnology sector has opened new avenues for enhancing the therapeutic potential of plant-based compounds. Nanoparticles, especially those synthesized from plant extracts, have shown great promise in improving the delivery and efficacy of anticancer agents. These plant-derived nanoparticles can target cancer cells more precisely, minimizing side effects and maximizing therapeutic outcomes. A chemical profiling study using UPLC-QTOF-MS/MS was conducted to explore the Portulaca oleracea (P. oleracea) phytoconstituents that exert the biological activities. Fifty compounds, comprising phenolic acids, organic acids, flavonoids, coumarins, fatty acids, alkaloids and amino acids were recognized via chemical characterization. The Folin.-Ciocalteu technique was utilized to assess the overall phenolics content of the obtained fractions, while the total amount of flavonoid was obtained using AlCl3. Moreover, the antioxidant ability was measured by ABTS, FRAP and DPPH techniques. Our findings revealed that the methanolic fraction has the greatest level of flavonoid (121.47 ±1.41 mg RE/g extract) and phenolic amounts (555.15 ± 1.14 mg GAE / g extract) among the examined extracts also the total alkaloid and the total saponin percent of the methanolic extract are 1.79% and 1.28% respectively. Moreover, methanol extract also show the highest antioxidant activity IC50 = 6.87, 21.31, 20.18µg/mL for ABTS, DPPH and FRAP respectively, this activity could be explained by the extract's high flavonoid and phenolics content. The water extract of P. oleracea was employed in producing gold nanoparticles AuNPs. UV-Vis spectrophotometric analysis and transmittance electron microscope (TEM) were used for analyzing the biosynthesized AuNPs. Additionally, the anti-cancer potential of the water extract and its biosynthesized AuNPs was explored by MTT assay against several cancer cell lines. The findings demonstrated that the the synthesized gold nanoparticles (AuNPs) was active against mammary gland breast cancer MCF-7 with an IC50 of (29.44±0.5) μg/mL compared to the reference medication, staurosporine STA with an IC50 of (12.24±0.68) μg/mL where in case of hepatocellular carcinoma HepG-2 cell lines the AuNPs has IC50 of (14.51±1.02) μg/mL, compared to staurosporine STA IC50 (5.20±0.87) μg/mL. The IC50valuesofAu-NPs were 31.14±0.87µg/mL for cervical cancer HeLa, 18.11±2.1 µg/mL for colon cancer cell lines Caco-2, 21.0±0.99 µg/mL, for HCT116 and17.47±1.3 µg/mL, for kidney cancer cell line UO-31 cell line. Across all cancer cell lines tested, the synthesized gold nanoparticles (AuNPs) from P. oleracea extract consistently demonstrated lower IC50 values compared to the crude water extract, indicating that the synthesis of AuNPs enhances the anticancer activity of the plant extract, this can be attributed to several factors, including their small size, which allows for better penetration into cancer cells, and their ability to induce reactive oxygen species (ROS), leading to apoptosis. The combination of plant-based bioactive compounds and nanotechnology provides a promising strategy for developing effective anticancer therapies.